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Abstract:

The present invention relates to compositions and methods of use thereof
for cancer therapy sensitization. Such compositions comprise functional
fragments of the nucleotide and/or polypeptide sequences of a Secreted
Protein Acidic and Rich in Cysteine (SPARC). The compositions can be used
in combination with existing chemotherapeutic agents for treatment of
cancers.

Claims:

72. The isolated polypeptide of claim 71, wherein the polypeptide
consists of SEQ ID NO: 3 and up to an additional 50 amino acids located
the amino or carboxyl terminus or both termini of SEQ ID NO: 3.

74. The isolated polypeptide of claim 71, wherein the polypeptide
consists of SEQ ID NO: 5 and up to an additional 50 amino acids located
the amino or carboxyl terminus or both termini of SEQ ID NO: 5.

76. The isolated polypeptide of claim 71, wherein the polypeptide
consists of SEQ ID NO: 7 and up to an additional 50 amino acids located
the amino or carboxyl terminus or both termini of SEQ ID NO: 7.

78. The isolated polypeptide of claim 71, wherein the polypeptide
consists of SEQ ID NO: 8 and up to an additional 50 amino acids located
the amino or carboxyl terminus or both termini of SEQ ID NO: 8.

80. The isolated polypeptide of claim 71, wherein the polypeptide
consists of SEQ ID NO: 9 and up to an additional 50 amino acids located
the amino or carboxyl terminus or both termini of SEQ ID NO: 9.

82. A composition comprising one or more of the isolated polypeptides of
any one of claims 1-11 and a pharmaceutically acceptable carrier.

83. The composition of claim 82, further comprising a cancer therapeutic
agent.

84. The composition of claim 83, wherein the cancer therapeutic agent is
selected from the group consisting of one or more chemotherapeutic
agents, one or more radiotherapeutic agents, one or more alternative
therapeutic agents, and combinations thereof.

Description:

[0001] The invention relates to cancer therapy sensitizing compositions
and methods, specifically polypeptides and polynucleotides relating to
the SPARC protein and the SPARC gene.

BACKGROUND OF THE INVENTION

[0002] Cancer is one of the leading causes of death in humans and while
standard chemotherapy, radiotherapy and surgical intervention
successfully reduce tumor load in many cases, resistance to
chemotherapeutic intervention is not uncommon, especially in solid
tumors. Resistance develops following exposure to chemotherapy and
further impedes tumor regression and cure. It is this chemotherapy
resistance leading to treatment failure that accounts for the high
mortality rates in cancer.

[0003] The molecular basis of chemotherapy resistance is largely genetic,
and can take many forms. Many mutations responsible for the initial
development of tumors may also contribute to drug resistance. For
example, loss of DNA mismatch repair (MMR) gene function has been
associated with a more rapid emergence of clinical drug resistance in
some cancers (de las Alas M. M., et al., 1997. J Natl Canc Inst
89:1537-41; Lin X. and Howell, S. B. 1999. Mol Pharmacol 56:390-5), and
mutations in the K-ras gene (found in approximately 40% of adenomatous
polyps and adenocarcinomas) are associated with an increased relapse
rate, mortality and a poor chemotherapeutic response (Arber N. et al.
2000. Gastroenterology 118:1045-1050). Aberrant expression and
dysregulation of proteins involved in the normally tightly regulated cell
replication cycle may also be protective of tumors--these proteins may be
loosely referred to as `oncogenes` in the literature. Gene products p21
and p27, for example, have been shown to protect tumors from undergoing
apoptosis elicited by various anticancer agents (Waldman T. et al., 1996.
Uncoupling of S phase and mistosis induced by anticancer agents in cells
lacking p21. Nature 381:713 716; St. Croix B. et al., 1996. Nature Med
1996, 2:1204-1210). Adhesion molecules, such as E-cadherin, may also
confer resistance to cells exposed to chemotherapeutic agents (Skoudy A,
et al., 1996. Biochem J 317: 279-84.). The mechanisms involved in
therapeutic resistance are varied and may be very complex.

[0004] Chemosensitizers may act in concert with the chemotherapeutic
agent, or may serve to counteract resistance mechanisms in the cell.
Existing chemosensitizers include small molecule drugs such as
photosensitizers or drug efflux pump inhibitors, and more recently,
antisense oligonucleotides. New compounds with chemosensitizing activity
include U.S. Pat. No. 5,776,925 and WO 02/00164, which provide examples
of novel chemical compounds that enhance cytotoxicity of therapeutic
agents.

[0005] U.S. Pat. No. 6,001,563 provides for a method for identifying
chemical compounds that may have chemosensitizing activity.

[0007] Similarly, cancer therapy sensitizers may act in concert with
cancer therapeutic agents, e.g., radiotherapy, or may serve to counteract
resistance mechanisms in the cell to the cancer therapeutic agent.

[0012] Some peptides corresponding to the cationic region of murine SPARC
and act as stimulators of capillary growth in vitro and in vivo. However,
Cu2+ binding activity alone does not appear to be sufficient for a
peptide to stimulate angiogenesis (Lane et al 1994. J Cell Biol
125:929-943).

[0015] The use of the intact, isolated SPARC protein as a chemosensitizer
is described by WO 2004/064785.

SUMMARY OF THE INVENTION

[0016] In accordance with one embodiment of the invention, there is
provided an isolated polynucleotide comprising the sequence of SEQ ID
NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:11 or SEQ ID NO:12, or an
isolated polypeptide comprising the sequence of SEQ ID NO: 3 or SEQ ID
NO: 5 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9. In a related
embodiment, the invention provides isolated polypeptides, wherein the
polypeptides have the amino acid sequence of any of SEQ ID NOs: 3, 5 or
7-10 and up to an additional 50 amino acids, preferably up to an
additional 25 amino acids, more preferably up to an additional 15 amino
acids, most preferably up to an additional 10 amino acids, wherein the
additional amino acids are located at the amino or carboxyl terminus or
both termini. The resulting polypeptides, made in accordance with the
invention, include polypeptides that are less than 50 amino acids in
total length. In a further related embodiment, the invention also
provides isolated polynucleotides which encode polypeptides having the
amino acid sequence of SEQ ID NOs: 3, 5 and 7-10 with additional amino
acids located at the amino or carboxyl terminus or both termini.

[0017] In accordance with another embodiment of the invention, there is
provided an isolated polypeptide selected from amino acids 17-153 of SEQ
ID NO:10, wherein the polypeptide has cancer therapeutic sensitizing
activity. In accordance with another embodiment of the invention, there
is provided an isolated polynucleotide selected from amino acids 157-56
of SEQ ID NO:10, wherein the polynucleotide has cancer therapeutic
sensitizing activity when expressed.

[0018] In accordance with another embodiment of the invention, there is
provided a medicament comprising SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ
ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. The invention may further
comprise a medicament comprising SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID
NO:7 or SEQ ID NO:8 or SEQ ID NO:9, and a chemotherapeutic agent. The
invention may further comprise a medicament comprising both SEQ ID NO: 3
or SEQ ID NO: 5 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9, in
combination with a cancer therapeutic agent, including, without
limitation, wherein the cancer therapeutic agent is selected from the
group consisting of one or more chemotherapeutic agents, one or more
radiotherapeutic agents, one or more alternative therapeutic agents, and
combinations thereof.

[0020] In accordance with another embodiment of the invention, there is
provided a method of sensitizing a cancerous cell to a cancer therapeutic
regimen, the method comprising; delivery of a vector comprising SEQ ID
NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO:11 or SEQ ID NO:12, to
a cell; expressing the sequence carried by the vector, and; treating the
cell with a chemotherapeutic agent.

[0021] In accordance with a related embodiment of the invention, there is
provided a method of sensitizing a cancerous cell to a cancer therapeutic
regimen, the method comprises; delivering a polypeptide comprising SEQ ID
NO: 3 or SEQ ID NO: 5 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9 to a
cell and thereafter treating the cell with a cancer therapeutic agent.
These methods of sensitizing cancerous cells may be practiced in
accordance with the invention, wherein the cancer therapeutic agent is
selected from the group consisting of one or more chemotherapeutic
agents, one or more radiotherapeutic agents, one or more alternative
therapeutic agents, and combinations thereof.

[0022] In accordance with another aspect of the invention, there is
provided a polypeptide comprising at least 5 consecutive amino acids of
SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID
NO:9. The polypeptide may further be combined or administered in
combination with a medicament comprising a chemotherapeutic agent.

[0023] In accordance with another aspect of the invention, there is
provided a medicament comprising; a polypeptide comprising at least 5
consecutive amino acids of SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO:7 or
SEQ ID NO:8 or SEQ ID NO:9, and a chemotherapeutic agent.

[0024] In accordance with another aspect of the invention, there is
provided a medicament comprising a polypeptide comprising at least 5
consecutive amino acids of SEQ ID NO: 8 or SEQ ID NO: 9, and a
chemotherapeutic agent. The invention may further comprise a medicament
comprising both SEQ ID NO: 8 and SEQ ID NO: 9, and a chemotherapeutic
agent.

[0025] In yet another aspect, the invention provides isolated sensitizing
polypeptides comprised of the sequences SEQ ID NOs: 3, 5 and 7-10,
wherein one or more amino acids has undergone a conservative mutation.
Also provided herein are isolated polynuceotides encoding said
conservatively mutated polypeptides.

[0026] In accordance with another embodiment of the invention, there is
provided a method of sensitizing a cancerous cell to a cancer therapeutic
agent, the method including: (a) delivering a polypeptide; selected from
one or more of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 8 and
SEQ ID NO: 9; to a cell; and (b) treating the cell with the cancer
therapeutic agent.

[0027] In accordance with another embodiment of the invention, there is
provided a method of sensitizing a cancerous cell to a cancer therapeutic
agent, the method including: (a) delivering a vector comprising a
polynucleotide selected from one or more of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO:6, SEQ ID NO: 11 and SEQ ID NO: 12; to a cell; (b) expressing
the sequence carried by the vector, and (c) treating the cell with a
cancer therapeutic agent.

[0028] In accordance with another embodiment of the invention, there is
provided a vector including an isolated polynucleotide selected from one
or more of the following: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ
ID NO: 11; SEQ ID NO: 12; and fragments, variants, or analogs thereof,
wherein the fragments, variants, or analogs thereof retain cancer
therapeutic sensitizing activity when expressed.

[0029] In accordance with another embodiment of the invention, there is
provided a cell including a polynucleotide described herein, wherein the
polynucleotide is operably linked to an expression control sequence.

[0030] In accordance with another embodiment of the invention, there is
provided a cell transfected with the vector as described herein or
progeny thereof.

[0031] In accordance with another embodiment of the invention, there is
provided a method of expressing a polypeptide, including: (a) providing
an expression vector encoding the polypeptide, wherein the polypeptide is
selected from one or more of the following: SEQ ID NO: 3; SEQ ID NO: 5;
SEQ ID NO:7; SEQ ID NO: 8; SEQ ID NO: 9; and fragments, variants, analogs
or conservatively mutated polypeptides thereof, wherein the fragments,
variants, analogs or conservatively mutated polypeptides thereof retain
cancer therapeutic sensitizing activity; (b) introducing the vector into
a cell; and (c) maintaining the cell under conditions permitting
expression.

[0032] The fragments, variants, analogs or conservatively mutated
polypeptides thereof, may be determined by one or more of the following:
% identity; % similarity; and the degree of conservation as described
herein.

[0033] The polynucleotide fragments, polynucleotide variants, or
polynucleotide analogs thereof, may be determined by one or more of the
following: % identity; % similarity; and the ability to hybridize under
highly stringent conditions, as described herein.

[0034] The delivering and the treating may be simultaneous. The delivering
may precede the treating or alternatively, the treating may precede the
delivering.

[0035] The cancer therapeutic agent may be selected from the group
consisting of one or more chemotherapeutic agents, one or more
radiotherapeutic agents, one or more alternative therapeutic agents, and
combinations thereof.

[0038] The cancerous cell may be selected on the basis that it is
resistant to a therapeutic regimen. The methods described herein may also
comprise a selection step for a cancerous cell that is resistant to a
therapeutic regimen.

[0041] The isolated polynucleotide may be operably linked to an expression
control sequence. The vector may be suitable for gene therapy.

[0042] The cell may be operable to express the polypeptides described
herein.

[0043] The introducing of the vector into a cell may be done in vivo. The
introducing of the vector into a cell may be done ex vivo. The
introducing of the vector into a cell may be done in vitro.

[0044] Other aspects and features of the present invention will become
apparent to those ordinarily skilled in the art upon review of the
following description of specific embodiments of the invention in
conjunction with the accompanying figures.

[0052]FIG. 7 shows the results of in vivo sensitization to the
chemotherapeutic agent 5-FU as measured by tumor growth in a xenograft
system comprising transplanted control tumor cells (growth with and
without 5-FU).

[0053]FIG. 8 shows the results of in vivo sensitization to the
chemotherapeutic agent 5-FU as measured by tumor growth in a xenograft
system comprising transplanted tumor cells expressing SEQ ID NO:
2/NT-Fragment (growth with and without 5-FU).

[0054]FIG. 9 shows the results of in vivo sensitization to the
chemotherapeutic agent 5-FU as measured by tumor growth in a xenograft
system comprising transplanted tumor cells expressing SEQ ID NO:
4/FS-Fragment (growth with and without 5-FU).

[0055]FIG. 10 shows the results of in vivo sensitization to the
chemotherapeutic agent 5-FU as measured by tumor growth in a xenograft
system comprising transplanted tumor cells expressing SEQ ID NO:
6/EC-Fragment (growth with and without 5-FU).

DETAILED DESCRIPTION

[0056] In the description that follows, a number of terms are used
extensively, the following definitions are provided to facilitate
understanding of the invention.

[0057] Current chemotherapy is limited by the ability of the patient to
tolerate the drug, and the ability of the cell to resist the cytotoxic
effects of the drug. Enhancing or mimicking the enhanced expression of a
protein with a tumor suppressive role in the normal cell may avoid the
toxicity issues of small molecules, and may have a longer effective
half-life when administered to the patient, enabling reduction of the
dosage while rendering cancerous cells more susceptible to the
chemotherapeutic agent.

[0058] As used herein, a "medicament" is a composition capable of
producing an effect that may be administered to a patient or test
subject. The effect may be chemical, biological or physical, and the
patient or test subject may be human, or a non-human animal, such as a
rodent or transgenic mouse. The composition may include small organic or
inorganic molecules with distinct molecular composition made
synthetically, found in nature, or of partial synthetic origin. Included
in this group are nucleotides, nucleic acids, amino acids, peptides,
polypeptides, proteins, peptide nucleic acids or complexes comprising at
least one of these entities. The medicament may be comprised of the
effective composition alone or in combination with a pharmaceutically
acceptable excipient.

[0059] As used herein, a "pharmaceutically acceptable excipient" includes
any and all solvents, dispersion media, coatings, antibacterial,
antimicrobial or antifungal agents, isotonic and absorption delaying
agents, and the like that are physiologically compatible. The excipient
may be suitable for intravenous, intraperitoneal, intramuscular,
intrathecal or oral administration. The excipient may include sterile
aqueous solutions or dispersions for extemporaneous preparation of
sterile injectable solutions or dispersion. Use of such media for
preparation of medicaments is known in the art.

[0060] As used herein, a "pharmacologically effective amount" of a
medicament refers to using an amount of a medicament present in such a
concentration to result in a therapeutic level of drug delivered over the
term that the drug is used. This may be dependent on the mode of
delivery, time period of the dosage, age, weight, general health, sex and
diet of the subject receiving the medicament. The determination of what
dose is a "pharmacologically effective amount" requires routine
optimization, which is within the capabilities of one of ordinary skill
in the art.

[0061] As used herein, the term "cancer" refers to a proliferative
disorder caused or characterized by the proliferation of cells which have
lost susceptibility to normal growth control. The term cancer, as used in
the present application, includes tumors and any other proliferative
disorders. Cancers of the same tissue type usually originate in the same
tissue, and may be divided into different subtypes based on their
biological characteristics. Four general categories of cancers are
carcinoma (epithelial tissue derived), sarcoma (connective tissue or
mesodermal derived), leukemia (blood-forming tissue derived) and lymphoma
(lymph tissue derived). Over 200 different types of cancers are known,
and every organ and tissue of the body may be affected. Specific examples
of cancers that do not limit the definition of cancer may include
melanoma, leukemia, astrocytoma, glioblastoma, retinoblastoma, lymphoma,
glioma, Hodgkins' lymphoma and chronic lymphocyte leukemia. Examples of
organs and tissues that may be affected by various cancers include
pancreas, breast, thyroid, ovary, uterus, testis, prostate, thyroid,
pituitary gland, adrenal gland, kidney, stomach, esophagus, colon or
rectum, head and neck, bone, nervous system, skin, blood, nasopharyngeal
tissue, lung, urinary tract, cervix, vagina, exocrine glands and
endocrine glands. Alternatively, a cancer may be multicentric or of
unknown primary site (CUPS).

[0062] As used herein, a `cancerous cell` refers to a cell that has
undergone a transformation event and whose growth is no longer regulated
to the same extent as before said transformation event. A tumor refers to
a collection of cancerous cells, often found as a solid or semi-solid
lump in or on the tissue or a patient or test subject.

[0063] A cancer or cancerous cell may be described as "sensitive to" or
"resistant to" a given therapeutic regimen or chemotherapeutic agent
based on the ability of the regimen to kill cancer cells or decrease
tumor size, reduce overall cancer growth (i.e. through reduction of
angiogenesis), and/or inhibit metastasis. Cancer cells that are resistant
to a therapeutic regimen may not respond to the regimen and may continue
to proliferate. Cancer cells that are sensitive to a therapeutic regimen
may respond to the regimen resulting in cell death, a reduction in tumor
size, reduced overall growth (tumor burden) or inhibition of metastasis.
For example, this desirably manifest itself in a reduction in tumor size,
overall growth/tumor burden or the incidence of metastasis of about 10%
or more, for example, about 30%, about 40%, about 50%, about 60%, about
70%, about 80%, or more, to about 2-fold, about 3-fold, about 4-fold,
about 5-fold, about 10-fold, about 15-fold, about 20-fold or more.
Monitoring of a response may be accomplished by numerous pathological,
clinical and imaging methods as described herein and known to persons of
skill in the art.

[0064] A common theme for a chemotherapeutic agent or combination of
agents is to induce death of the cancerous cells. For example, DNA
adducts such as nitrosoureas, busulfan, thiotepa, chlorambucil,
cisplatin, mitomycin, procarbazine, or dacacarbazine slow the growth of
the cancerous cell by forcing the replicating cell to repair the damaged
DNA before the M-phase of the cell cycle, or may by themselves cause
sufficient damage to trigger apoptosis of the cancerous cell. Other
events such as gene expression or transcription, protein translation, or
methylation of the replicated DNA, for example, may also be interfered
with by the varied arsenal of chemotherapeutic agents available to the
clinician and help to trigger apoptotic processes within the cancerous
cells. Alternately, a chemotherapeutic agent may enable the cancerous
cell to be killed by aspects of the patient or test subject's humoral or
acquired immune system, for example, the complement cascade or lymphocyte
attack.

[0065] While not desiring to be bound by any specific theories, a
cancerous cell resistant to a chemotherapeutic agent or combination of
agents may fight for its survival by actively transporting the drug out
of the cell for example, by overexpression of the ABC transporter MDR1
p-glycoprotein (FORD et al 1993. Cytotechnol. 12: 171-212) or acquiring
`counter-mutations` to counteract the drugs. For example, mutations in
the DNA repair enzymes that affect the ability to detect damage to the
cells' DNA may enable replication of the damaged DNA and permit the
cancerous cells to continue replicating, enlarging the tumor. As
mutations accumulate, other regulatory points that would otherwise act in
a normal cell cycle cease to function, and the cycle of unregulated
growth cascades. Another aspect of chemotherapeutic resistance involves
the tumor cells' avoidance of apoptosis. A host organism's normal
response to dysregulated cell growth is to initiate apoptosis and
eliminate the defective cell before the cascade into uncontrolled
replication begins. However, this may be subverted by a cancerous cell,
for example, by disruption of signal transduction events, loss of
adhesion dependence or contact inhibition in the cancerous cell, or loss
of apoptosis-promoting factors, often considered `tumor suppressors`, for
example p53, BRCA1 or RB. The importance of this sensitivity to apoptosis
in the treatment of cancer is supported by recent evidence indicating
that the selectivity of chemotherapy for the relatively few tumors ever
cured solely by drugs depends, to a large extent, upon their easy
susceptibility to undergo apoptosis (JOHNSTONE et al., 2002. Cell.
108(2):153-64).

[0066] As used herein, a "therapeutic regimen" or "therapy" refers to the
administration of at least one agent which is harmful to cancerous cells.
Suitable therapeutic regimens for use in accordance with the invention
include, but are not limited to, "chemotherapeutic regimens,"
"radiotherapeutic regimens," "alternative therapeutic regimen" and
combinations thereof.

[0067] As used herein, a "chemotherapeutic regimen" or "chemotherapy"
refers to the administration of at least one chemotherapy agent which is
harmful to destroy cancerous cells. There are a myriad of such
chemotherapy agents available to a clinician. Chemotherapy agents may be
administered to a subject in a single bolus dose, or may be administered
in smaller doses over time. A single chemotherapeutic agent may be used
(single-agent therapy) or more than one agent may be used in combination
(combination therapy). Chemotherapy may be used alone to treat some types
of cancer. Alternatively, chemotherapy may be used in combination with
other types of treatment, for example, radiotherapy or alternative
therapies (for example immunotherapy) as described herein. Additionally,
a chemosensitizer may be administered as a combination therapy with a
chemotherapy agent.

[0068] As used herein, a "chemotherapeutic agent" refers to a medicament
that may be used to treat cancer, and generally has the ability to kill
cancerous cells directly. Examples of chemotherapeutic agents include
alkylating agents, antimetabolites, natural products, hormones and
antagonists, and miscellaneous agents. Examples of alternate names are
indicated in brackets. Examples of alkylating agents include nitrogen
mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan
(L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such
as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan;
nitrosoureas such as carmustine (BCNU), semustine (methyl-CCNU),
lomustine (CCNU) and streptozocin (streptozotocin); DNA synthesis
antagonists such as estramustine phosphate; and triazines such as
dacarbazine (DTIC, dimethyl-triazenoimidazolecarboxamide) and
temozolomide. Examples of antimetabolites include folic acid analogs such
as methotrexate (amethopterin); pyrimidine analogs such as fluorouracin
(5-fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR),
cytarabine (cytosine arabinoside) and gemcitabine; purine analogs such as
mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG)
and pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine and
fludarabine; and topoisomerase inhibitors such as amsacrine. Examples of
natural products include vinca alkaloids such as vinblastine (VLB) and
vincristine; taxanes such as paclitaxel and docetaxel (Taxotere);
epipodophyllotoxins such as etoposide and teniposide; camptothecins such
as topotecan and irinotecan; antibiotics such as dactinomycin
(actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin,
bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin; enzymes such
as L-asparaginase; and biological response modifiers such as interferon
alpha and interlelukin 2. Examples of hormones and antagonists include
luteinising releasing hormone agonists such as buserelin;
adrenocorticosteroids such as prednisone and related preparations;
progestins such as hydroxyprogesterone caproate, medroxyprogesterone
acetate and megestrol acetate; estrogens such as diethylstilbestrol and
ethinyl estradiol and related preparations; estrogen antagonists such as
tamoxifen and anastrozole; androgens such as testosterone propionate and
fluoxymesterone and related preparations; androgen antagonists such as
flutamide and bicalutamide; and gonadotropin-releasing hormone analogs
such as leuprolide. Examples of miscellaneous agents include thalidomide;
platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin
and carboplatin; anthracenediones such as mitoxantrone; substituted ureas
such as hydroxyurea; methylhydrazine derivatives such as procarbazine
(N-methylhydrazine, MIH); adrenocortical suppressants such as mitotane
(o,p'-DDD) and aminoglutethimide; RXR agonists such as bexarotene; and
tyrosine kinase inhibitors such as imatinib. Alternate names and
trade-names of these and additional examples of chemotherapeutic agents,
and their methods of use including dosing and administration regimens,
will be known to a person versed in the art, and may be found in, for
example "The Pharmacological basis of therapeutics", 10th edition.
HARDMAN H G., LIMBIRD L E. editors. McGraw-Hill, New York, and in
"Clinical Oncology", 3rd edition. Churchill Livingstone/Elsevier
Press, 2004. ABELOFF, M D. editor. In particular, suitable
chemotherapeutic agents for use in accordance with the invention include,
without limitation, nanoparticle albumin-bound paclitaxels.

[0069] As used herein, the term "radiotherapeutic regimen" or
"radiotherapy" refers to the administration of radiation to kill
cancerous cells. Radiation interacts with various molecules within the
cell, but the primary target, which results in cell death is the
deoxyribonucleic acid (DNA). However, radiotherapy often also results in
damage to the cellular and nuclear membranes and other organelles. DNA
damage usually involves single and double strand breaks in the
sugar-phosphate backbone. Furthermore, there can be cross-linking of DNA
and proteins, which can disrupt cell function. Depending on the radiation
type, the mechanism of DNA damage may vary as does the relative biologic
effectiveness. For example, heavy particles (i.e. protons, neutrons)
damage DNA directly and have a greater relative biologic effectiveness.
Electromagnetic radiation results in indirect ionization acting through
short-lived, hydroxyl free radicals produced primarily by the ionization
of cellular water. Clinical applications of radiation consist of external
beam radiation (from an outside source) and brachytherapy (using a source
of radiation implanted or inserted into the patient). External beam
radiation consists of X-rays and/or gamma rays, while brachytherapy
employs radioactive nuclei that decay and emit alpha particles, or beta
particles along with a gamma ray.

[0070] Radiotherapy may further be used in combination chemotherapy, with
the chemotherapeutic agent acting as a radiosensitizer. The specific
choice of radiotherapy suited to an individual patient may be determined
by a skilled person at the point of care, taking into consideration the
tissue and stage of the cancer.

[0073] In particular, suitable alternative therapeutic regimens include,
without limitation, antibodies to molecules on the surface of cancer
cells such as antibodies to Her2 (e.g., Trastuzumab), EGF or EGF
Receptors, VEGF (e.g., Bevacizumab) or VEGF Receptors, CD20, and the
like. The therapeutic agent may further comprise any antibody or antibody
fragment which mediates one or more of complement activation, cell
mediated cytotoxicity, inducing apoptosis, inducing cell death, and
opsinization. For example, such an antibody fragment may be a complete or
partial Fc domain.

[0074] By "antibodies" it is meant without limitation, monoclonal
antibodies, polyclonal antibodies, dimers, multimers, multispecific
antibodies (e.g., bispecific antibodies). Antibodies may be murine,
human, humanized, chimeric, or derived from other species. An antibody is
a protein generated by the immune system that is capable of recognizing
and binding to a specific antigen. A target antigen generally has
numerous binding sites, also called epitopes, recognized by CDRs on
multiple antibodies. Each antibody that specifically binds to a different
epitope has a different structure. Thus, one antigen may have more than
one corresponding antibody.

[0075] An antibody includes a full-length immunoglobulin molecule or an
immunologically active portion of a full-length immunoglobulin molecule,
i.e., a molecule that contains an antigen binding site that
immunospecifically binds an antigen of a target of interest or part
thereof. Targets include, cancer cells or other cells that produce
autoimmune antibodies associated with an autoimmune disease.

[0076] The immunoglobulins disclosed herein can be of any class (e.g.,
IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2) of immunoglobulin molecule. The immunoglobulins can be
derived from any species.

[0077] "Antibody fragments" comprise a portion of a full length antibody,
which maintain the desired biological activity. "Antibody fragments" are
generally the antigen binding or variable region thereof. Examples of
antibody fragments include Fab, Fab', F(ab')2, and Fv fragments;
diabodies; linear antibodies; fragments produced by a Fab expression
library, anti-idiotypic (anti-Id) antibodies, CDR (complementary
determining region), and epitope-binding fragments of any of the above
which immunospecifically bind to cancer cell antigens, viral antigens or
microbial antigens, single-chain antibody molecules; and multispecific
antibodies formed from antibody fragments.

[0078] The monoclonal antibodies referenced herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light chain
is identical with or homologous to corresponding sequences in antibodies
derived from a particular species or belonging to a particular antibody
class or subclass, while the remainder of the chain(s) is identical with
or homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass, as
well as fragments of such antibodies, so long as they exhibit the desired
biological activity (U.S. Pat. No. 4,816,567). Chimeric antibodies of
interest herein include "primatized" antibodies comprising variable
domain antigen-binding sequences derived from a non-human primate (e.g.,
Old World Monkey or Ape) and human constant region sequences.

[0079] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to
a cell-mediated reaction in which nonspecific cytotoxic cells that
express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells,
neutrophils, and macrophages) recognize bound antibody on a target cell
and subsequently cause lysis of the target cell. The primary cells for
mediating ADCC, NK cells, express Fc.γ.RIII only, whereas monocytes
express FcγRI, FcγRII and FcγRIII. To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay may be
performed (U.S. Pat. No. 5,003,621; U.S. Pat. No. 5,821,337). Useful
effector cells for such assays include peripheral blood mononuclear cells
(PBMC) and Natural Killer (NK) cells. Alternatively, or additionally,
ADCC activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al PNAS (USA),
95:652-656 (1998).

[0080] An antibody which "induces cell death" is one which causes a viable
cell to become nonviable. Cell death in vitro may be determined in the
absence of complement and immune effector cells to distinguish cell death
induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC). Thus, the assay for cell death
may be performed using heat inactivated serum (i.e., in the absence of
complement) and in the absence of immune effector cells. To determine
whether the antibody is able to induce cell death, loss of membrane
integrity as evaluated by uptake of propidium iodide (PI), trypan blue or
7AAD can be assessed relative to untreated cells. Cell death-inducing
antibodies are those which induce PI uptake in the PI uptake assay in
BT474 cells.

[0081] An antibody which "induces apoptosis" is one which induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum,
cell fragmentation, and/or formation of membrane vesicles (called
apoptotic bodies).

[0082] As used herein, a "chemosensitizer" or "sensitizer" is a medicament
that may enhance the therapeutic effect of a chemotherapeutic agent,
radiotherapy treatment or alternative therapeutic regimen, and therefore
improve efficacy of such treatment or agent. The sensitivity or
resistance of a tumor or cancerous cell to treatment may also be measured
in an animal, such as a human or rodent, by, e.g., measuring the tumor
size, tumor burden or incidence of metastases over a period of time. For
example, about 2, about 3, about 4 or about 6 months for a human and
about 2-4, about 3-5, or about 4-6 weeks for a mouse. A composition or a
method of treatment may sensitize a tumor or cancerous cell's response to
a therapeutic treatment if the increase in treatment sensitivity or the
reduction in resistance is about 10% or more, for example, about 30%,
about 40%, about 50%, about 60%, about 70%, about 80%, or more, to about
2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about
15-fold, about 20-fold or more, compared to treatment sensitivity or
resistance in the absence of such composition or method. The
determination of sensitivity or resistance to a therapeutic treatment is
routine in the art and within the skill of a person versed in the art.

[0083] The terms "peptide," "polypeptide," and "protein" may be used
interchangeably, and refer to a compound comprised of at least two amino
acid residues covalently linked by peptide bonds or modified peptide
bonds, for example peptide isosteres (modified peptide bonds) that may
provide additional desired properties to the peptide, such as increased
half-life. A peptide may comprise at least two amino acids. The amino
acids comprising a peptide or protein described herein may also be
modified either by natural processes, such as posttranslational
processing, or by chemical modification techniques which are well known
in the art. Modifications can occur anywhere in a peptide, including the
peptide backbone, the amino acid side-chains and the amino or carboxyl
termini. It is understood that the same type of modification may be
present in the same or varying degrees at several sites in a given
peptide.

[0085] A substantially similar sequence is an amino acid sequence that
differs from a reference sequence only by one or more conservative
substitutions as discussed herein. Such a sequence may, for example, be
functionally homologous to another substantially similar sequence. It
will be appreciated by a person of skill in the art the aspects of the
individual amino acids in a peptide of the invention that may be
substituted.

[0086] Amino acid sequence similarity or identity may be computed by,
e.g., using the BLASTP and TBLASTN programs which employ the BLAST (basic
local alignment search tool) 2.0 algorithm. Techniques for computing
amino acid sequence similarity or identity are well known to those
skilled in the art, and the use of the BLAST algorithm is described in
ALTSCHUL et al. 1990, J. Mol. Biol. 215: 403-410 and ALTSCHUL et al.
(1997), Nucleic Acids Res. 25: 3389-3402.

[0088] Alignments of protein sequences may be conducted using existing
algorithms to search databases for sequences similar to a query sequence.
One alignment method is the Smith-Waterman algorithm (Smith, T. F. and
Waterman, M. S. 1981. Journal of Molecular Biology 147(1):195-197), which
is useful in determining how an optimal alignment between the query
sequence and a database sequence can be produced. Such an alignment is
obtained by determining what transformations the query sequence would
need to undergo to match the database sequence. Transformations include
substituting one character for another and inserting or deleting a string
of characters. A score is assigned for each character-to-character
comparison-positive scores for exact matches and some substitutions,
negative scores for other substitutions and insertions/deletions. Scores
are obtained from statistically-derived scoring matrices. The combination
of transformations that results in the highest score is used to generate
an alignment between the query sequence and database sequence. The
Needleman-Wunsch (Needleman, S. B. and Wunsch, C. D. 1970. Journal of
Molecular Biology 48(3):443-453) algorithm is similar to the
Smith-Waterman algorithm, but sequence comparisons are global, not local.
Global comparisons force an alignment of the entire query sequence
against the entire database sequence. While local alignments always begin
and end with a match, global alignments may begin or end with an
insertion or deletion (indel). For a given query sequence and database
sequence, a global score will be less than or equal to a local score due
to indels on the ends. As an alternative to the above algorithms, a
Hidden Markov Model (HMM) search (Eddy, S. R. 1996. Current Opinion in
Structural Biology 6(3):361-365) could be used to generate protein
sequence alignments. HMM scoring weighs the probability of a match being
followed by insertions/deletions or vice-versa. In addition, HMMs allow
insertion to deletion transitions (and vice versa) and scoring of begin
and end states to control whether a search is run globally or locally.

[0089] One or more of the above algorithms may be used in an alignment
program to generate protein sequence alignments. A person skilled in the
art has numerous sequence alignment programs to choose from, that
incorporate a variety of different algorithms. One example of an
alignment program is BLASTP (Altschul, S. F., et al. (1997) Nucleic Acids
Res. 25(17):3389-3402). Other alignment programs are CLUSTAL W and
PILEUP. The standard output from a BLASTP run contains enough information
to conduct further indel analysis as described below.

[0090] Amino acids may be described as, for example, polar, non-polar,
acidic, basic, aromatic or neutral. A polar amino acid is an amino acid
that may interact with water by hydrogen bonding at biological or
near-neutral pH. The polarity of an amino acid is an indicator of the
degree of hydrogen bonding at biological or near-neutral pH. Examples of
polar amino acids include serine, proline, threonine, cysteine,
asparagine, glutamine, lysine, histidine, arginine, aspartate, tyrosine
and glutamate. Examples of non-polar amino acids include glycine,
alanine, valine leucine, isoleucine, methionine, phenylalanine, and
tryptophan. Acidic amino acids have a net negative charge at a neutral
pH. Examples of acidic amino acids include aspartate and glutamate. Basic
amino acids have a net positive charge at a neutral pH. Examples of basic
amino acids include arginine, lysine and histidine. Aromatic amino acids
are generally nonpolar, and may participate in hydrophobic interactions.
Examples of aromatic amino acids include phenylalanine, tyrosine and
tryptophan. Tyrosine may also participate in hydrogen bonding through the
hydroxyl group on the aromatic side chain. Neutral, aliphatic amino acids
are generally nonpolar and hydrophobic. Examples of neutral amino acids
include alanine, valine, leucine, isoleucine and methionine. An amino
acid may be described by more than one descriptive category. Amino acids
sharing a common descriptive category may be substitutable for each other
in a peptide.

[0091] Nomenclature used to describe the peptide compounds of the present
invention follows the conventional practice where the amino group is
presented to the left and the carboxy group to the right of each amino
acid residue. In the sequences representing selected specific embodiments
of the present invention, the amino- and carboxy-terminal groups,
although not specifically shown, will be understood to be in the form
they would assume at physiologic pH values, unless otherwise specified.
In the amino acid structure formulae, each residue may be generally
represented by a one-letter or three-letter designation, corresponding to
the trivial name of the amino acid, in accordance with the following
Table 1:

[0093] Amino acids comprising the peptides described herein will be
understood to be in the L- or D-configuration. In peptides and
peptidomimetics of the present invention, D-amino acids may be
substitutable for L-amino acids.

[0094] Amino acids contained within the peptides of the present invention,
and particularly at the carboxy-or amino-terminus, may be modified by
methylation, amidation, acetylation or substitution with other chemical
groups which may change the circulating half-life of the peptide without
adversely affecting their biological activity. Additionally, a disulfide
linkage may be present or absent in the peptides of the invention.

[0095] Nonstandard amino acids may occur in nature, and may or may not be
genetically encoded. Examples of genetically encoded nonstandard amino
acids include selenocysteine, sometimes incorporated into some proteins
at a UGA codon, which may normally be a stop codon, or pyrrolysine,
sometimes incorporated into some proteins at a UAG codon, which may
normally be a stop codon. Some nonstandard amino acids that are not
genetically encoded may result from modification of standard amino acids
already incorporated in a peptide, or may be metabolic intermediates or
precursors, for example. Examples of nonstandard amino acids include
4-hydroxyproline, 5-hydroxylysine, 6-N-methyllysine,
gamma-carboxyglutamate, desmosine, selenocysteine, ornithine, citrulline,
lanthionine, 1-aminocyclopropane-1-carboxylic acid, gamma-aminobutyric
acid, carnitine, sarcosine, or N-formylmethionine. Synthetic variants of
standard and non-standard amino acids are also known and may include
chemically derivatized amino acids, amino acids labeled for
identification or tracking, or amino acids with a variety of side groups
on the alpha carbon. Examples of such side groups are known in the art
and may include aliphatic, single aromatic, polycyclic aromatic,
heterocyclic, heteronuclear, amino, alkylamino, carboxyl, carboxamide,
carboxyl ester, guanidine, amidine, hydroxyl, alkoxy, mercapto-,
alkylmercapto-, or other heteroatom-containing side chains. Other
synthetic amino acids may include alpha-imino acids, non-alpha amino
acids such as beta-amino acids, des-carboxy or des-amino acids. Synthetic
variants of amino acids may be synthesized using general methods known in
the art, or may be purchased from commercial suppliers, for example RSP
Amino Acids LLC (Shirley, Mass.).

[0096] In order to further exemplify what is meant by a conservative amino
acid substitution, Groups A-F are listed below. The replacement of one
member of the following groups by another member of the same group is
considered to be a conservative substitution.

[0097] Group A includes leucine, isoleucine, valine, methionine,
phenylalanine, serine, cysteine, threonine, and modified amino acids
having the following side chains: ethyl, iso-butyl, --CH2CH2OH,
--CH2CH2CH2OH, --CH2CHOHCH3 and
CH2SCH3.

[0098] Group B includes glycine, alanine, valine, serine, cysteine,
threonine, and a modified amino acid having an ethyl side chain.

[0099] Group C includes phenylalanine, phenylglycine, tyrosine,
tryptophan, cyclohexylmethyl, and modified amino residues having
substituted benzyl or phenyl side chains.

[0102] Group F includes serine, threonine, cysteine, and modified amino
acids having C1-C5 straight or branched alkyl side chains substituted
with --OH or --SH.

[0103] Groups A-F are exemplary and are not intended to limit the
invention.

[0104] A peptidomimetic is a compound comprising non-peptidic structural
elements that mimics the biological action of a parent peptide. A
peptidomimetic may not have classical peptide characteristics such as an
enzymatically scissile peptidic bond. A parent peptide may initially be
identified as a binding sequence or phosphorylation site on a protein of
interest, or may be a naturally occurring peptide, for example a peptide
hormone. Assays to identify peptidomimetics may include a parent peptide
as a positive control for comparison purposes, when screening a library,
such as a peptidomimetic library. A peptidomimetic library is a library
of compounds that may have biological activity similar to that of a
parent peptide.

[0105] As used herein, the term "polynucleotide" includes RNA, cDNA,
genomic DNA, synthetic forms, and mixed polymers, both sense and
antisense strands, and may be chemically or biochemically modified or may
contain non-natural or derivatized nucleotide bases, as will be readily
appreciated by those skilled in the art. Such modifications include, for
example, labels, methylation, substitution of one or more of the
naturally occurring nucleotides with an analog, internucleotide
modifications such as uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages
(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties
(e.g., polypeptides), and modified linkages (e.g., alpha anomeric
polynucleotides, etc.). Also included are synthetic molecules that mimic
polynucleotides in their ability to bind to a designated sequence via
hydrogen bonding and other chemical interactions.

[0106] "Peptide nucleic acids" (PNA) as used herein refer to modified
nucleic acids in which the sugar phosphate skeleton of a nucleic acid has
been converted to an N-(2-aminoethyl)-glycine skeleton. Although the
sugar-phosphate skeletons of DNA/RNA are subjected to a negative charge
under neutral conditions resulting in electrostatic repulsion between
complementary chains, the backbone structure of PNA does not inherently
have a charge. Therefore, there is no electrostatic repulsion.
Consequently, PNA has a higher ability to form double strands as compared
with conventional nucleic acids, and has a high ability to recognize base
sequences. Furthermore, PNAs are generally more robust than nucleic
acids. PNAs may also be used in arrays and in other hybridization or
other reactions as described above and herein for oligonucleotides.

[0107] As used herein, the term "vector" refers to a polynucleotide
compound used for introducing exogenous or endogenous polynucleotide into
host cells. A vector comprises a nucleotide sequence, which may encode
one or more polypeptide molecules. Plasmids, cosmids, viruses and
bacteriophages, in a natural state or which have undergone recombinant
engineering, are non-limiting examples of commonly used vectors to
provide recombinant vectors comprising at least one desired isolated
polynucleotide molecule.

[0108] As used herein, a "tumor suppressor" is a gene or gene product that
has a normal biological role of restraining unregulated growth of a cell.
If the function of a tumor suppressor is lost, unregulated cell growth
arises. The functional counterpart to a tumor suppressor is an
oncogene--genes that promote normal cell growth may be known as
`protooncogenes`. A mutation that activates such a gene or gene product
further converts it to an `oncogene`, which continues the cell growth
activity, but in a dysregulated manner. Examples of tumor suppressor
genes and gene products are well known in the literature and may include
PTC, BRCA1, BRCA2, p16, APC, RB, WT1, EXT1, p53, NF1, TSC2, NF2, VHL or
SPARC. Further examples of tumor suppressor genes, gene products and
their functions may be found in: McKusick, V. A.: Mendelian Inheritance
in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns
Hopkins University Press, 1998 (12th edition), and in the online
companion site: Online Mendelian Inheritance in Man, OMIM®.
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University
(Baltimore, Md.) and National Center for Biotechnology Information,
National Library of Medicine (Bethesda, Md.), 2000. World Wide Web URL:
http://www.ncbi.nlm.nih.gov/omim/.

[0109] The invention further provides nucleic acid constructs comprising
control elements and a nucleic acid molecule described herein operatively
linked to the control elements (e.g., a suitable promoter) for expression
of a polypeptide or a polypeptide herein described. Protein expression is
dependent on the level of RNA transcription, which is in turn regulated
by DNA signals. Similarly, translation of mRNA requires, at the very
least, an AUG initiation codon, which is usually located within about 10
to about 100 nucleotides of the 5' end of the message. Sequences flanking
the AUG initiator codon have been shown to influence its recognition by
eukaryotic ribosomes, with conformity to a perfect Kozak consensus
sequence resulting in optimal translation (see, e.g., Kozak, J. Molec.
Biol. 196: 947-950 (1987)). Also, successful expression of an exogenous
nucleic acid in a cell can require post-translational modification of a
resultant protein. Accordingly, the invention provides plasmids encoding
polypeptides wherein the vector is, e.g., pcDNA3.1 or a derivative
thereof.

[0110] The nucleic acid molecules described herein preferably comprise a
coding region operatively linked to a suitable promoter, which promoter
is preferably functional in eukaryotic cells. Viral promoters, such as,
without limitation, the RSV promoter and the adenovirus major late
promoter can be used in the invention. Suitable non-viral promoters
include, but are not limited to, the phosphoglycerokinase (PGK) promoter
and the elongation factor 1α promoter. Non-viral promoters are
desirably human promoters. Additional suitable genetic elements, many of
which are known in the art, also can be ligated to, attached to, or
inserted into the inventive nucleic acid and constructs to provide
additional functions, level of expression, or pattern of expression. The
native promoters for expression of the SPARC family genes also can be
used, in which event they are preferably not used in the chromosome
naturally encoding them unless modified by a process that substantially
changes that chromosome. Such substantially changed chromosomes can
include chromosomes transfected and altered by a retroviral vector or
similar process. Alternatively, such substantially changed chromosomes
can comprise an artificial chromosome such as a HAC, YAC, or BAC.

[0111] In addition, the nucleic acid molecules described herein may be
operatively linked to enhancers to facilitate transcription. Enhancers
are cis-acting elements of DNA that stimulate the transcription of
adjacent genes. Examples of enhancers which confer a high level of
transcription on linked genes in a number of different cell types from
many species include, without limitation, the enhancers from SV40 and the
RSV-LTR. Such enhancers can be combined with other enhancers which have
cell type-specific effects, or any enhancer may be used alone.

[0112] To optimize protein production the inventive nucleic acid molecule
can further comprise a polyadenylation site following the coding region
of the nucleic acid molecule. Also, preferably all the proper
transcription signals (and translation signals, where appropriate) will
be correctly arranged such that the exogenous nucleic acid will be
properly expressed in the cells into which it is introduced. If desired,
the exogenous nucleic acid also can incorporate splice sites (i.e.,
splice acceptor and splice donor sites) to facilitate mRNA production
while maintaining an inframe, full length transcript. Moreover, the
inventive nucleic acid molecules can further comprise the appropriate
sequences for processing, secretion, intracellular localization, and the
like.

[0113] The nucleic acid molecules can be inserted into any suitable
vector. Suitable vectors include, without limitation, viral vectors.
Suitable viral vectors include, without limitation, retroviral vectors,
alphaviral, vaccinial, adenoviral, adenoassociated viral, herpes viral,
and fowl pox viral vectors. The vectors preferably have a native or
engineered capacity to transform eukaryotic cells, e.g., CHO-K1 cells.
Additionally, the vectors useful in the context of the invention can be
"naked" nucleic acid vectors (i.e., vectors having little or no proteins,
sugars, and/or lipids encapsulating them) such as plasmids or episomes,
or the vectors can be complexed with other molecules. Other molecules
that can be suitably combined with the inventive nucleic acids include
without limitation viral coats, cationic lipids, liposomes, polyamines,
gold particles, and targeting moieties such as ligands, receptors, or
antibodies that target cellular molecules.

[0114] One measure of "correspondence" of nucleic acids, peptides or
proteins for use herein with reference to the above described nucleic
acids and proteins is relative "identity" between sequences. In the case
of peptides or proteins, or in the case of nucleic acids defined
according to a encoded peptide or protein correspondence includes a
peptide having at least about 50% identity, alternatively at least about
70% identity, alternatively at least about 90% identity, or even about
95% and may also be at least about 98-99% identity to a specified peptide
or protein. Preferred measures of identity as between nucleic acids is
the same as specified above for peptides with at least about 90% or at
least about 98-99% identity being most preferred.

[0115] The term "identity" as used herein refers to the measure of the
identity of sequence between two peptides or between two nucleic acids
molecules. Identity can be determined by comparing a position in each
sequence, which may be a line for purposes of comparison. Two amino acid
or nucleic acid sequences are considered substantially identical if they
share at least about 75% sequence identity, preferably at least about 90%
sequence identity and even more preferably at least 95% sequence identity
and most preferably at least about 98-99% identity.

[0116] Sequence identity may be determined by the BLAST algorithm
currently is use and which was originally described in Altschul et al.
(1990) J. Mol. Biol. 215:403-410. The BLAST algorithm may be used with
the published default settings. When a position in the compared sequence
is occupied by the same base or amino acid, the molecules are considered
to have shared identity at that position. The degree of identity between
sequences is a function of the number of matching positions shared by the
sequences.

[0117] An alternate measure of identity of nucleic acid sequences is to
determine whether two sequences hybridize to each other under low
stringency, and preferably high stringency conditions. Such sequences are
substantially identical when they will hybridize under high stringency
conditions. Hybridization to filter-bound sequences under low stringency
conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium
dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in
0.2×SSC/0.1 SDS at 42° C. (see Ausubel et al. (eds.) 1989,
Current Protocols in Molecular Biology, Vol. 1, Green Publishing
Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3).
Alternatively, hybridization to filter-bound sequences under high
stringency conditions, may for example, be performed in 0.5 M
NaHPO4, 7% (SDS), 1 mM EDTA at 65° C., and washing in
0.2×SSC/0.1% SDS at 68° C. (see Ausubel et al. (eds.) 1989,
supra). Hybridization conditions may be modified in accordance with known
methods depending on the sequence of interest (see Tijssen, 1993,
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2
"Overview of Principles in Hybridization and the Strategy of Nucleic Acid
Probe Assays", Elsevier, N.Y.). Generally, stringent conditions are
selected to be about 5° C. lower than the thermal melting point
for the specific sequence at a defined ionic strength and pH.

[0118] The full-length SPARC protein has multiple functional domains,
including an N-terminal acidic domain, a follistatin-like domain that may
inhibit cell proliferation and a C-terminal extracellular domain that
binds calcium ions with high affinity and inhibits cell proliferation.
When the full-length SPARC protein is administered to a mouse tumour
model, chemosensitizing activity has been demonstrated, improving the
response rate of resistant tumours to chemotherapeutic agents, for
example 5-fluorouracil, irinotecan, cisplatin or etoposide.

[0119] Which of these domains, or regions within these domains, are
responsible for the apoptotic-mediating or chemosensitizing effects of
SPARC have not been previously investigated.

[0120] It will be appreciated by a person of skill in the art that the
numerical designations of the positions of mutations within a sequence
are relative to the specific sequence. Also the same positions may be
assigned different numerical designations depending on the way in which
the sequence is numbered and the sequence chosen. Furthermore, sequence
variations such as insertions or deletions, may change the relative
position and subsequently the numerical designations of particular
nucleotides at and around a mutational site.

[0123] Gene therapy is a medical intervention that involves modifying the
genetic material of living cells to fight disease. Gene therapy is being
studied in clinical trials (research studies with humans) for many
different types of cancer and for other diseases. Accordingly, the
invention further provides for an isolated nucleic acid molecule encoding
a SPARC polypeptide suitable for use in "gene therapy" (see, e.g., Patil
et al., AAPS J. 7(1):E61-77 (2005)).

[0124] In general, a gene is delivered to the cell using a "vector" such
as those disclosed herein. The most common types of vectors used in gene
therapy are viruses. Viruses used as vectors in gene therapy are
genetically disabled; they are unable to reproduce themselves. Most gene
therapy clinical trials rely on mouse retroviruses to deliver the desired
gene. Other viruses used as vectors include adenoviruses,
adeno-associated viruses, poxviruses, and the herpes virus. Suitable
viral gene therapy vectors and modes of their administration in vivo and
ex vivo are known in the art.

[0125] Gene therapy can be performed both ex vivo and in vivo. Typically,
in ex vivo gene therapy clinical trials, cells from the patient's blood
or bone marrow are removed and grown in the laboratory. The cells are
exposed to the virus that is carrying the desired gene. The virus enters
the cells, and the desired gene becomes part of the cells' DNA. The cells
grow in the laboratory and are then returned to the patient by injection
into a vein. Using in vivo gene therapy, vectors such as, e.g., viruses
or liposomes may be used to deliver the desired gene to cells inside the
patient's body.

[0126] Identification of key SPARC polypeptide sequences involved in
control of apoptosis precedes the development of peptide-based
chemosensitizing therapeutics to be used in conjunction with
chemotherapeutic agents. Individual synthetic polypeptides spanning at
least 5 consecutive amino acids of the N-terminal third of SPARC,
including the cleaved secretion signal sequence in the immature SPARC,
may, in combination with a chemotherapeutic agent, serve to sensitize the
cancerous cells in a resistant tumor. Assays to identify suitable
synthetic polypeptides may be performed by exposing resistant cancerous
cells in vitro or in vivo to at least one isolated polypeptide and a
chemotherapeutic agent (alone or in combination), followed by continued
exposure to a chemotherapeutic agent as described herein. Cell survival
and apoptosis may be assessed using methods described herein.

[0127] Peptides according to one embodiment of the invention may include
peptides comprising the amino acid sequences provided in Table 2. Other
peptides according to other embodiments of the invention may include
peptides having a substantially similar sequence to those provided in
Table 2. Such peptides may be in isolation, or may be linked to or in
combination with tracer compounds, protein translocation sequences,
liposomes, carbohydrate carriers, polymeric carriers or other agents or
excipients as will be apparent to one of skill in the art. In an
alternate embodiment, such peptides may comprise a medicament, wherein
such peptides may be present in a pharmacologically effective amount.

[0128] The present invention provides compositions and methods for
sensitizing cancer therapeutic treatments. Such sensitizing compositions
and methods are particularly useful in enhancing the response of patients
who are resistant to a treatment. They are also useful in reducing the
side-effects of cancer therapy, for example, by enhancing the response of
a patient to a smaller strength (i.e., dosage) of the treatment. The
composition of the present invention may reduce the dosage of a
therapeutic treatment agent by at least about 10%, for example, at least
about 30%, at least about 40%, at least about 50%, and at least about
60%.

[0129] Those of ordinary skill in the art will recognize that, because of
the universality of the genetic code, the knowledge of any given amino
acid sequence allows those of ordinary skill in the art to readily
envision a finite number of specific polynucleotide sequences that can
encode a polypeptide of said amino acid sequence. Further, the ordinarily
skilled artisan can readily determine the optimal polynucleotide sequence
to encode a polypeptide of said amino acid sequence for expression in any
given species via the process of "codon optimization," which is well know
in the art (see, e.g., VILLALOBOS et al.: Gene Designer: a synthetic
biology tool for constructing artificial DNA segments. BMC
Bioinformatics. 2006 Jun. 6; 7:285).

[0130] The present invention provides, in one embodiment, a composition
comprising a SPARC polypeptide and a chemotherapy-resistant cell. In
addition to sensitizing a sample or a mammal to cancer therapy, the use
of the subject compositions of the present invention can reduce the
dosage of a therapy, therefore reducing the side effects caused by cancer
therapy. The above compositions may comprise a pharmaceutical
composition, which further includes a pharmaceutically acceptable carrier
or excipient.

[0131] As used herein, a "carrier" refers to any substance suitable as a
vehicle for delivering an Active Pharmaceutical Ingredient (API) to a
suitable in vitro or in vivo site of action. As such, carriers can act as
an excipient for formulation of a therapeutic or experimental reagent
containing an API. Preferred carriers are capable of maintaining an API
in a form that is capable of interacting with a T cell. Examples of such
carriers include, but are not limited to water, phosphate buffered
saline, saline, Ringer's solution, dextrose solution, serum-containing
solutions, Hank's solution and other aqueous physiologically balanced
solutions or cell culture medium. Aqueous carriers MAY also contain
suitable auxiliary substances required to approximate the physiological
conditions of the recipient, for example, enhancement of chemical
stability and isotonicity. Suitable auxiliary substances include, for
example, sodium acetate, sodium chloride, sodium lactate, potassium
chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate,
and other substances used to produce phosphate buffer, Tris buffer, and
bicarbonate buffer.

[0136] Sensitive MIP 101 and resistant MIP/5FU cells were seeded at a
density of 8,000 cells/well (96-well plate) in DMEM supplemented with 10%
FBS, 1% penicillin/streptomycin. After incubation at 37° C. with
5% CO2 for 48 hrs, the media was removed and fresh serum-free
conditioned medium (VP-SFM, Invitrogen) containing the peptide of
interest was incubated for an additional 48 hrs in the presence or
absence of 500 μM 5-FU. Cell viability was assessed by MTS assay.

[0137] Peptides were custom synthesized by Sigma. Peptides A, A1, A2, A3
were dissolved in 0.1% DMSO. Peptides B8, B14, B8-scramble and
B14-scramble were dissolved in PBS. For the MTS cell viability assay, a
dose response curve was obtained using a "high" peptide concentration of
200 μg/ml, and a "low" peptide concentration of 96 μg/ml.

Cell Viability (MTS Assay)

[0138] For the MTS assay,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium inner salt (MTS) and phenazine methosulfate (PMS),
purchased from Promega (Madison, Wis.), were mixed in the proportions
2:0.92 μg/ml, and 25 μl of the mixture was added to each well.
After incubation at 37° C. with 5% CO2 for 2 h, the
absorbance of each well at 490 nm was measured in a 96-well plate reader
(Versa Max, version 4.8, Molecular Devices Co.) according to the
manufacturer's instructions. Cell viability was expressed as a percentage
of control.

[0139] Statistical difference between groups was determined by analysis of
variance followed by post hoc comparison with Student's t-test. A
P-value<0.05 was considered statistically significant

EXAMPLES

Example 1

[0140] Sensitive (MIP101--FIG. 1) and multi-drug resistant (MIP/CPT--FIG.
2) cells were transiently transfected and assayed for survival upon
exposure to irinotecan (CPT). Cell viability was quantified by an MTS
assay. Transfection of both SEQ ID NO: 2 and SEQ ID NO: 4 had a similar
effect by decreasing cell viability in the resistant MIP/CPT cells
compared to that of the full length SPARC vector. Resistant MIP/CPT cells
transfected with control (empty vector) remained viable despite exposure
to 500 uM CPT-11.

Example 2

Use of SPARC Peptide Chemosensitizers In Vitro

[0141] This example demonstrates the utility of the inventive SPARC
peptide chemosensitizers in an in vitro model system.

[0142] Shorter peptides corresponding to various regions of either the
N-terminal acidic region or the follistatin domain were synthesized to
further narrow the peptide sequences demonstrating chemosensitizing
activity. MIP101 sensitive or resistant to 5-FU (MIP/5FU) cells were
exposed to the B14 peptide (SEQ ID NO. 9) as described. In both resistant
and sensitive cells, the peptide alone, at either of the concentrations
tested did not significantly affect viability (FIG. 4). As expected the
resistant cells exhibited greater viability in the presence of 5-FU
alone--with the addition of the B 14 peptide at 96 or 200 μg/ml, cell
viability decreased compared to 5-FU alone, indicating that the presence
of the peptide increases the sensitivity of the resistant cells to the
chemotherapeutic agent.

[0143] A similar trend is observed when the MIP101 cells sensitive or
resistant to 5-FU (MIP/5FU) were exposed to the B8 peptide (SEQ ID NO.8)
(FIG. 3). The peptide alone, at either concentration did not affect cell
viability. In the resistant cells (MIP/5FU), the drug by itself had no
effect on cell viability, but in the presence of increasing
concentrations of peptide, there was increasing sensitivity of the
resistant cells to the chemotherapeutic agent.

[0145] This example demonstrates A utility of the inventive SPARC peptide
chemosensitizers in a tumor xenograft model system.

[0146] Tumor xenograft animal models were used to assess the effect of
overexpressing different SPARC fragments on tumor progression in vivo.
MIP 101 cells with stable transfection and expression of SPARC (MIP/SP)
or different biological fragments representing the N-terminus (MIP/NT),
follistatin (MIP/FS) and extracellular (MIP/EC) domains of SPARC were
used for the tumor xenograft model. NIH nude mice (6 weeks old, Taconic
Laboratories, Germantown, N.Y.) were implanted with 1×106
cells at the left flank. Treatment regimens were initiated once the
average tumor size reached 75-100 mm3 in volume. Tumors were
measured using a hand-held caliper (Fisher Scientific International, Inc.
Hampton, N.H.) with concurrent body weight measurements until the
completion of the study. Chemotherapy was provided using a 3-week cycle
regimen (×6 cycles in total): 5-FU 25 mg/kg body weight
intraperitoneal (IP) injections three times on week 1 of each cycle,
followed by 2 weeks of treatment-free periods. Control animals received
saline injections. Each group contained 6-8 animals.

[0147] The results are shown in FIGS. 7-10. MIP 101 cells stably
expressing the N-terminus (NT-fragment; SEQ ID NO: 2) and extracellular
(EC-fragment; SEQ ID NO: 6) domains of SPARC demonstrated significant
sensitization to 5-FU in this xenotransplant system. Specifically, the
NT-fragment appears to provide the greatest sensitivity in vivo, followed
by the EC-fragment, and lastly, the FS-fragment. The EC-domain shows a
delayed moderate chemosensitizing effect, which occurs after about 44
days of treatment in the present assay.

[0148] While specific embodiments of the invention have been described and
illustrated, such embodiments should be considered illustrative of the
invention only and not as limiting the invention.